Development of a stable low-copy mini F plasmid derivative for evaluating β-lactamase substrate specificity through antimicrobial susceptibility testing

Traditionally, the impact of the substrate specificity of β-lactamase produced by bacteria on β-lactam resistance (β-lactamase-mediated resistance) has been assessed by performing antimicrobial susceptibility testing (AST) on transformants carrying the cloned gene on arbitrarily chosen β-lactamase-free plasmid vectors. However, because the promoter strength and plasmid copy number of these vectors are not standardized, the resulting β-lactamase expression levels vary, further hampering cross-study comparisons. We constructed pMiniF-1 by inserting a tac promoter, a ribosome-binding site, and transcriptional terminators into the mini F plasmid, which is maintained at one to two copies per cell. This design enables controlled β-lactamase expression for accurate evaluation of β-lactamase-mediated resistance via AST. This approach enables the determination and comparison of MICs with β-lactamase expression levels controlled to be as nearly identical as possible. We performed AST on Escherichia coli DH5αΔblaEC-like transformants expressing 22 β-lactamase genes (Ambler classes A-D), including carbapenemases that were cloned into pMiniF-1. The resulting antimicrobial susceptibility profiles and MIC differences did not contradict the known substrate specificities of the enzymes. Meropenem and imipenem MICs for the IMP-6 producer were fourfold higher and eightfold lower, respectively, than IMP-1. Aztreonam MICs were similar between transformants lacking β-lactamase genes and those with class B β-lactamases. Imipenem MIC for the OXA-48 producer was ≥8-fold higher than for OXA-1, whereas cefepime MICs were 16-fold lower. We demonstrate that pMiniF-1 enables the comparison of β-lactamase substrate specificity via AST. The pMiniF-1 plasmid (Catalog No. RDB21122) is available from the RIKEN BioResource Research Center (Tsukuba, Ibaraki, Japan) (https://web.brc.riken.jp/en/).IMPORTANCEβ-lactamases play a central role in bacterial resistance to β-lactam antibiotics, and novel variants continue to emerge. However, the impact of individual β-lactamase variants on β-lactam resistance across diverse enzyme-substrate combinations remains insufficiently characterized. Traditional kinetic analyses require enzyme purification and multiple reaction assays, which hinder comprehensive experimental evaluation. Expression-based approaches have also been used to assess β-lactamase function, but conventional systems depend on unstable high-copy plasmids, raising concerns about reproducibility due to variable plasmid copy numbers. In this study, we developed pMiniF-1, a stable, low-copy plasmid that enables consistent gene expression without antibiotic selection, thereby maintaining stable copy numbers under drug-free conditions. Using 22 β-lactamase genes, we demonstrated that susceptibility testing with pMiniF-1 adequately reflects their known substrate specificity profiles. This system provides a reliable method for assessing β-lactamase substrate specificity and will facilitate comparative studies on the functional diversity and evolution of emerging β-lactamase variants.